This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The choice of probes for clinical use is primarily organic fluorophores because of their favorable toxicity and pharmacokinetic properties. Additionally, organic dyes can be easily conjugated to peptides or other functional groups that will bind to cancer cell receptors. Organic fluorophores may also be tagged with a delivery vehicle that has high affinity to a particular cell in question. The development of these fluorescent probes facilitates diagnostic molecular imaging, and the specific and prolonged uptake of these fluorophores in the body is modulated by designing optimum chemical, physical and thus disease-specific properties. Accordingly, when a fluorescent dye is conjugated to a biological entity the photophysical properties of that dye may change. Therefore the development of these NIR organic dye constructs begins with in vitro diagnostics prior to small animal injection and clinical translation. Approach NIR fluorophores often have overlapping spectra either with each other or with various chromophores found in vivo. Autofluorescence in the NIR contributes to increased background noise and reduced target-to-background measurements. When designing a fluorescent probe it is thus important to select one with optimum spectral properties. The high-resolution spectral system will be used to (i) measure spectra and compare similar organic dyes to select the optimum fluorophore, (ii) distinguish between labeled species on the cell for measuring binding mechanisms, and (iii) examine fluorescent species that are dual-labeled for multi-wavelength molecular imaging. Additionally, the phase-sensitive flow system will be used to (i) measure the lifetime of fluorescent constructs when bound to cell surfaces for comparison to the un-bound probe, (ii) detect phase-sensitive NIR fluorescence and discriminate that from cellular autofluorescence on fluorophore bound cells, and (iii) compare the lifetimes of competitive cell-bound fluophores exhibiting similar emission spectra.